Urine Biomarker Discovery for the Early Detection of Liver Cancer. Urine contains macro-molecules that can be used for the early detection of diseases and cancers that occur at non-urinary tract sites. Given the many advantages of using urine in biomarker monitoring, the goal of this project is to develop systems for the biomarker discovery of macromolecules (DNA and glycan) in urine that may ultimately improve the prognosis for such a disease. Comparative analysis of DNA modifications and glycan species (host and, where appropriate, viral) will be performed using urine samples from individuals at different stages of health and disease up to the diagnosis of liver cancer. This project will thus deliver new methods and new biomarkers for HCC and ultimately other cancers using urine as the biological fluid. These goals will be achieved by the following Aims. Aim 1: Detection of genetically or epigenetically modified DNA markers that have previously been associated with the development of HCC in urine as potential urine biomarkers for the early detection of HCC. The assays for detecting genetic and epigenetic modifications associated with HCC will be developed for analyzing urine DNA. The detection of each DNA marker in the urine of patients with different stages of liver disease and healthy subjects as the control will be performed to evaluate each marker as a candidate for a HCC, urine-derived biomarker panel. Aim 2: Determine if free glycan, isolated from human urine, can be used for biomarker discovery and the early detection of HCC. This Aim will determine if uncomplex glycans (presumably remnants of glycoproteins) present in urine can be a source for cancer biomarkers. The species of glycan, isolated from the urine of individuals, as a function of disease stage will be comparatively analyzed by HPLC. Glycan structures that appear to correlate with a diagnosis of liver disease will be characterized, and will be further explored as possible biomarkers. The promising urine-derived biomarkers obtained from Aim 1 and 2 will be used to propose another phase of study in which a larger scale, pre-validation study can be instituted to determine the sensitivity and specificity of each biomarker and the predicative value of a single biomarker or a combination of biomarkers for the early detection of HCC. This knowledge can ultimately be used so that a panel of multiple urine-derived biomarkers for the early detection of HCC can be constructed. Upon accomplishment of this project, we will have developed the technology platform for biomarker discovery that uses urine as the biological sample fluid, which can then be applied to other cancers. PUBLIC HEALTH RELEVANCE: This proposal aims to discover biomarkers for the early detection of liver cancer using urine as the biological sample fluid. The use of urine would be both appropriate and advantageous because urine can contain biomarkers from non- urinary tract sites, and the use of urine would be non-invasive. Upon accomplishment of this project, we will have developed the technology platform for biomarker discovery in urine, which can then be applied to other cancers.